1. Field of the Invention
The present invention relates to a method for preparing the endocrine aggregate (EA) of insulin-producing beta cells from human pluripotent stem cells (hPSCs).
2. Description of the Related Art
Stem cells are the cells in the phase right before the differentiation into each cell constructing each organ, which have self renewal capacity that makes unlimited proliferation in the non-differentiated state possible and have pluripotency, the ability to be differentiated into various tissues by a specific differentiation stimulus. That is, even after continued culture, self renewal capacity of the cells does not decrease and rather stays in order to make differentiation into various cells possible.
Stem cells are largely divided into two types, which are embryonic stem cells (ESCs) and adult stem cells (ASCs), according to the differentiation potential. When a sperm meets an egg, this leads to fertilization, followed by development and morphogenesis. At this time, the cells are through proliferation, migration, and differentiation processes. Embryonic stem cells are the cells separated from the inner cell mass (ICM) to be developed into a fetus, among the very beginning stage blastocyst embryos, before the fertilized egg is implanted in the endometrium, which are the pluripotent cells generated in three embryonic germ layers such as endoderm, ectoderm, and mesoderm to be differentiated into every cell to form every tissue.
In the meantime, adult stem cells are the organ specific stem cells obtained from the placenta in the stage of organ formation in the embryo under development or from an adult who has already been through all the cell development. The differentiation potency of these stem cells is limited in tissue specific cells, indicating that these cells are multipotent. Adult stem cells remain in most organs after all the differentiation to be grown up finishes in order to supplement any loss caused either normally or pathogenically. The representative adult stem cells are hematopoietic stem cells existing in the bone marrow and mesenchymal stem cells in the middle of differentiation into connective tissue except blood cells. Hematopoietic stem cells are differentiated into various blood cells including erythrocytes and leucocytes, while mesenchymal stem cells are differentiated into osteoblasts, chondroblasts, adipocytes, and myoblasts, etc.
Ever since the separation of human embryonic stem cells succeeded, the interest in their clinical application has been increased. Cell replacement therapy using stem cells as the perfect cell supplier has also been in the spotlight. Parkinson's disease, one of intractable diseases, neurodegenerative disease such as Alzheimer's disease, quadriplegia caused by spinal cord injury, leukemia, stroke, juvenile diabetes, myocardial infarction, and liver cirrhosis are such diseases that are caused by the destruction and permanent malfunction of tissue forming cells. Cell replacement therapy is the method to replace and supply cells for those diseases demonstrating the lack of cells.
Diabetes Mellitus is the disease that causes various acute/chronic complications and as a result it might cause various diseases and disorders in patients, resulting in early incompetence and even early death. This disease causes the increase of social medical costs and causes a deal of loss of labor, resulting in putting more burdens on the society overall. According to the World Health Report made by WHO in 1997, it is presumed that the number of diabetic patients is going to be increased to approximately 300 million in 2025. Especially, many developing countries in Asia and Africa will face diabetes epidemic carried by westernized way of life including diet. According to the epidemiologic study of diabetes in Korea, the prevalence rate of diabetes in Korean people at the age of over 30 s is presumed to be 8˜9%, indicating the rate is rapidly increasing along with the modernization of society.
The treatment of diabetes mostly depends on insulin therapy. Oral hypoglycemic agent, insulin secretagogue, and insulin sensitizer are also used along with diet therapy and exercise therapy. However, modern medicine cannot cure the disease completely and transplantation of pancreatic islets might be the fundamental treatment, which though has problems of absolute lack of donors and side effects accompanied by the continuous administration of immunosuppressants.
The study on embryonic stem cells to treat diabetes has been actively going on, in which the study on the differentiation of pluripotent cells into insulin producing cells having the function of pancreatic β-cells stands in the middle. Lumelsky, et al. reported the differentiation of mouse embryonic stem cells into islet cells like insulin producing structure (Science 292:1389, 2001). Hori, et al. reported that when the insulin producing cells obtained from the mouse embryonic stem cells prepared by adding Pl-3 kinase suppressor were administered to the mouse diabetic model induced by streptozotocin, the insulin producing cells could regulate the high blood sugar level to the normal (PNAS 99:16105, 2002). In 1998, Thomson, et al. established human embryonic stem cells from the human blastula. Afterwards, the attempts have been actively made to obtain human insulin producing cells by differentiating the established human embryonic stem cells. However, it has been disclosed that human embryonic stem cells have unique stem cell characteristics and are very much different from mouse embryonic stem cells in their signal transduction system necessary for the cell differentiation into mature cells. As an attempt to prepare human insulin producing cells from human embryonic stem cells, Assady, et al. screened insulin producing cells by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) (Diabetes 50:1691, 2001). Later, the same research group reported that they succeeded in establishing insulin producing cells by taking advantage of the differentiation method proposed by Lumelsky, et al. (Stem Cells 22:265, 2004). However, the limitation in insulin production and secretion could not be overcome, yet.
Moreover, most insulin observed by immunocytochemistry in previous papers were the ones originated from culture media rather than biosynthesized in cells, that is cells absorbed insulin from the culture fluid during the culture and then later released it back to the medium by stimuli including high glucose level. Researchers even insisted that the insulin producing cells at this time were not healthy ones with experiencing apoptosis and many agreed with that (Sipione, et al., Diabetologia 47:499, 2004; Hansson, et al., Diabetes 53:2603, 2004; Rajagopal, et al., Science 299:363, 2003). Therefore, it is common understanding that the previous methods to produce insulin described in papers and patent documents have to be modified and further a novel method for inducing cell differentiation and identification thereof has to be studied. In particular, for the application to the clinical use, high purity insulin producing cells or cell clusters having normal functions have to be provided, asking continuous study to establish an efficient differentiation method. Considering the above, the patent technology retained by Geron Cooperation (10-2004-7008713, PCT/US2002/039089) has a weakness. That is, Geron Cooperation did not prove that the insulin observed by immunocytochemistry in the final product obtained by the patent technology was biosynthesized endogenously. Besides, no proof was included in that invention to explain whether or not the insulin therein was released out of the cells by an external glucose stimulus. In that patent technology, the differentiation was induced by using a cocktail comprising various differentiation factors having every possibility to affect the differentiation in their ways, but the analysis on such affection by those factors was not provided clearly.
In the course of study, to overcome the above problems, on the differentiation and proliferation mechanism of human embryonic stem cells into insulin producing beta cells, the present inventors succeeded in producing insulin secreting endocrine cells by inducing the differentiation of human embryonic stem cells or human induced pluripotent stem cells into definitive endoderm (DE), pancreatic endoderm (PE), endocrine progenitors (EP), and endocrine cells (EC) stepwise in that order. Particularly, the present inventors confirmed the conditions for the formation of the endocrine aggregate (EA) from the said endocrine cells, and confirmed that the said endogenous aggregate (EA) has the cell proliferation potential at a significant level, leading to the completion of this invention.